Redesign of the proton-pumping machinery of cytochrome c oxidase: Proton pumping does not require Glu(I-286)

One of the putative proton-transfer pathways leading from solution toward t he binuclear center in many cytochrome c oxidases is the D-pathway, so-call ed because it starts with a highly conserved aspartate [D(I-132)] residue. Another highly conserved amino acid residue in this pathway, glutamate(I-28 6), has been indicated to play a central role in the proton-pumping machine ry of mitochondrial-type enzymes, a role that requires a movement of the si de chain between two distinct positions. In the present work we have reloca ted the glutamate to the opposite side of the proton-transfer pathway by co nstructing the double mutant EA(I-286)/IE(I-112). This places the side chai n in about the same position in space as in the original enzyme, but does n ot allow for the same type of movement. The results show that the introduct ion of the second-site mutation, IE(I-112), in the EA(I-286) mutant enzyme results in an increase of the enzyme activity by a factor of >10. In additi on, the double mutant enzyme pumps similar to0.4 proton per electron. This observation restricts the number of possible mechanisms for the operation o f the redox-driven proton pump. The proton-pumping machinery evidently does require the presence of a protonatable/polar residue at a specific locatio n in space, presumably to stabilize an intact water chain. However, this re sidue does not necessarily have to be at a strictly conserved location in t he amino acid sequence. In addition, the results indicate that E(I-286) is not the "proton gate" of cytochrome c oxidase controlling the flow of pumpe d protons from one to the other side of the membrane.